Difference between revisions of "Part:BBa K2889001:Design"

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The secondary structure of IL7-AS
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The secondary structure of IL7-AS(Fig 4).
 
https://static.igem.org/mediawiki/parts/f/f7/IL7-AS.jpg
 
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Revision as of 12:36, 9 October 2018


pSB1C3-IL7-AS


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2194
    Illegal PstI site found at 167
    Illegal PstI site found at 581
    Illegal PstI site found at 612
    Illegal PstI site found at 735
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1529
    Illegal PstI site found at 167
    Illegal PstI site found at 581
    Illegal PstI site found at 612
    Illegal PstI site found at 735
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2194
    Illegal PstI site found at 167
    Illegal PstI site found at 581
    Illegal PstI site found at 612
    Illegal PstI site found at 735
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2194
    Illegal PstI site found at 167
    Illegal PstI site found at 581
    Illegal PstI site found at 612
    Illegal PstI site found at 735
    Illegal AgeI site found at 1375
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 127
    Illegal BsaI.rc site found at 1183
    Illegal SapI.rc site found at 429


Design Notes

IL7-AS [located anti-sense to interleukin-7 (Il7) gene; Accession number: NM_000880.3] is a newly discovered lncRNA that can be induced across multiple human and mouse cell types and has been reported to regulate the expression of interleukin-6 (IL6). We want to investigate the funtion of IL7-AS in kidney renal clear cell carcinoma. We cloned the full length of IL7-AS (3079bp) and inserted into pSB1C3 for submitting to IGEM 2018. We also inserted the full length of IL7-AS into pCDNA3.1 to study it's function in kidney renal clear cell carcinoma associated cell lines.

1.1 Amplification of IL7-AS fragments in human cells.

First we amplified IL7-AS fragment(line 2) using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with EcoRI and PstI (Fig 1).

Amplification_of_IL7-AS_and_IL7-AS-S2.jpeg


1.2 Digested PSB1C3 vector. We digested the PSB1C3 vectors with EcoRI and PstI (Fig 2).

Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg

1.3 Ligation of purified IL7-AS fragments to pSB1C3 vector. IL7-AS fragments were ligated to pSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3).

Verify_pSB1C3-IL7-AS_by_sequencing.jpeg

The secondary structure of IL7-AS(Fig 4). IL7-AS.jpg

Source

We cloned the full length of IL-AS from RNA of human cells.

References